Bacteriological examinations
In this study, the preliminary clinical examination of the mammary glands of 3240 dairy goats indicated that 55 (1.7%) goats with clinical mastitis, of which 8/55 (14.5%) were primiparous and 47/55 (85.5%) were multiparous. Of the 55 goats, 43 (78.18%) goats had only one udder half affected.
We analyzed 55 milk samples collected from 55 dairy goats with clinical mastitis. Ultimately, one hundred and forty-nine bacteria isolates were identified and divided into fifty-two different bacterial species, and Staphylococcus appeared in each milk sample, and it was the predominant species as it accounted for 36.91% (55/149) of the identified isolates, followed by Enterobacter 15.44% (23/149), Proteus 12.08% (18/149), Klebsiella 10.07% (15/149), and others 25.5% (38/149). Among the 55 Staphylococcus isolates, S. aureus (n = 26) was the predominant as it isolated in 26 samples, followed by S. haemolyticus (n = 10), S. epidermidis (n = 6), S. saprophyticus (n = 5), S. gallinarum (n = 4), S. galilis (n = 1), S. coni (n = 1), S. simulans (n = 1), and S. equorum (n = 1). While the Streptococcus was not isolated in this study.
Antimicrobial susceptibility profile
The antimicrobial resistant phenotype of fifty-five Staphylococcus isolates from dairy goat mastitis to 9 antimicrobial agents were showed in Table 1. The tested Staphylococcus isolates were resistance to all tested β-lactam antibiotics (penicillin, ampicillin, and ceftriaxone). S. aureus and S. haemolyticus were sensitive to amikacin, clindanlycin, ciprofloxacin, acetylisovale-ryltylosin and doxycycline.More than 60% of S. epidermidis, S. saprophyticus and S. gallinarum were resistance to the tested antimicrobial agents, and most of them expressed the MDR phenotype. S. galilis, S. coni, S. simulans and S. equorum were resistance to all tested antimicrobial agents.
Pathogenicity of the isolated bacteria
The S. aureus isolates were used in the pathogenicity experiment as it was the most prevalent bacteria based on the bacteriological examinations. We observed the general performance of the tested mice in order to know the effects of S. aureus on clinical symptoms of mice. We found that the majority of mice infected with S. aureus exhibited clinical signs, including mental depression, slow response, lethargy, reduced spontaneous activity and anorexia,. The mice of the tested group began to die after two days. After 7 days, the survival rate of mice in the control group was 100%, whereas the fatality rate in the tested group was 80%.
Histopathological examinations of the main organs of mice in tested group and negative control group were performed. In the control group, the liver, heart, lung and kidney tissues of mice exhibited normal structure (The Histopathological images were not shown). Compared to the control group, obvious pathological changes were observed in the liver, kidney and lung tissues of the mice in the tested group. Necrotic foci were observed in liver, some hepatocytes occurred the cytoplasmic vacuoles and karyopyknosis. Moreover, some hepatocytes were reduced in size, with hyperchromatic nuclei (Fig. 1A). For kidney tissues, renal tubular epithelial cells fused, the nucleolus disappeared, and separated from the basement membrane of the renal tubule. The cellular necrosis and neutrophil infiltration were seen in the glomerular capillary network (Fig. 1B). Alveolar walls fused, and the alveolar space was filled with numerous inflammatory exudates, neutrophils and lymphocytes (Fig. 1C). However, no significant pathological changes were observed in the heart tissue of infected mice, except the nucleolus of the some myocardial cells disappeared, and the striated muscle disappeared (Fig. 1D).
The antimicrobial activities of the Tibetan herbal medicines
The in vitro antibacterial activities of the water extracts of six Tibetan herbal medicines against the Staphylococcus isolates were showed in Fig. 2, Table 1 and Table 2 based on the Kirby-Bauer disc diffusion and microdilution methods. The results indicated that the six Tibetan herbal medicines exhibited various degrees of antimicrobial activity, and the inhibition zone diameter ranged from 7.0 to 25.5 mm. Among the tested Staphylococcus isolates, the larger inhibition zone diameter (Ø 25.5mm) was against S. aureus and the smallest (Ø 7.0 mm) was against S. epidermidis and S. coni. In the comparative study, the water extracts of Lagotis brachystachy exhibited the best antibacterial effect to all tested Staphylococcus isolates, followed by Aconitum flavum, 89.1% (49/55) of Staphylococcus isolates were highly sensitive to Aconitum flavum. Others, 98.2% (54/55), 89.1% (49/55) and 83.6% (46/55) of Staphylococcus isolates were moderately sensitive to Uncaria rhynchophylla, Swertia bimaculata, and Dracocephalum tanguticum, respectively. While the water extracts of Gentiana urnula showed the weakest antibacterial activity to Staphylococcus isolates.
Table 1
The susceptibility of 55 Staphylococcus isolates from dairy goat mastitis to 9 antimicrobial agents
Isolates
|
Minimal inhibitory concentration (MIC, μg/mL)
|
Penicillin
|
Amoxicillin
|
Ceftriaxone
|
Ceftiofur
|
Amikacin
|
Clindanlycin
|
Ciprofloxacin
|
Acetylisovale-ryltylosin
|
Doxycycline
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
S
|
I
|
R
|
≤0.5
|
1
|
≥2.0
|
≤0.5
|
1
|
≥2.0
|
≤2
|
4
|
≥8
|
≤2
|
4
|
≥8
|
≤16
|
32
|
≥64
|
≤0.5
|
1-2
|
≥4
|
≤0.5
|
1-2
|
≥4
|
≤0.5
|
1-4
|
≥8
|
≤0.5
|
8
|
≥16
|
S. aureus(n=26)
|
8-32
|
4-32
|
8-32
|
1-8
|
0.125-16
|
8-32
|
0.125-8
|
0.125-8
|
0.125-4
|
S. haemolyticus(n=10)
|
8-32
|
8-32
|
8-64
|
1-16
|
0.125-8
|
1-16
|
2-8
|
0.25-8
|
0.125-2
|
S. epidermidis(n=6)
|
16-64
|
8-64
|
16-128
|
2-32
|
16-128
|
2-8
|
16-64
|
2-16
|
8-256
|
S. saprophyticus(n=5)
|
4-32
|
16-64
|
16-256
|
2-32
|
1-256
|
4-32
|
2-64
|
1-16
|
64-256
|
S. gallinarum(n=4)
|
4-32
|
8-32
|
16-128
|
4-128
|
1-64
|
8-32
|
1-32
|
4-16
|
4-256
|
S. galilis(n=1)
|
16
|
16
|
64
|
16
|
0.5
|
4
|
128
|
8
|
4
|
S. coni(n=1)
|
32
|
32
|
64
|
32
|
128
|
32
|
64
|
16
|
256
|
S. simulans(n=1)
|
256
|
>256
|
128
|
128
|
>256
|
>256
|
64
|
8
|
256
|
S. equorum(n=1)
|
16
|
16
|
16
|
32
|
4
|
8
|
8
|
2
|
64
|
ATCC 25923 (n=1)
|
0.5
|
0.125
|
0.5
|
0.25
|
0.125
|
0.125
|
0.5
|
0.25
|
0.5
|
Note: "S" means sensitive; "I" means intermediate; "R" means resistant.
|
Table 2
The results of inhibition zone of six Tibetan medicinal plants against Staphylococcus isolates
Isolates
|
Inhibition zone diameter(mm)
|
Lagotis
brachystachy
|
Aconitum flavum
|
Uncaria
rhynchophylla
|
Swertia bimaculataaa
|
Dracocephalum tanguticum
|
Gentiana
urnula
|
Lonicera japonica
|
S. aureus (n = 26)
|
19.3–25.5
|
15.1–20.3
|
13.3–18.3
|
12.2–15.3
|
12.5–15.7
|
9.1–13.9
|
14.4–18.0
|
S. haemolyticus(n = 10)
|
18.2–24.2
|
16.2–19.8
|
13.5–16.4
|
10.0-15.8
|
9.3–14.7
|
8.3–12.5
|
13.2–17.8
|
S. epidermidis(n = 6)
|
19.2–25.3
|
14.6–19.4
|
11.1–17.3
|
10.2–16.9
|
11.3–13.5
|
7.3–10.0
|
12.0-16.5
|
S. saprophyticus(n = 5)
|
16.2–24.3
|
14.2–18.8
|
11.3–16.0
|
10.2–14.3
|
10.2–12.6
|
9.4–11.5
|
12.3–17.7
|
S. gallinarum(n = 4)
|
18.1–24.0
|
14.4–17.6
|
10.4–15.6
|
9.4–14.6
|
10.4–13.6
|
8.2–10.8
|
11.4–15.3
|
S. galilis(n = 1)
|
21.5
|
18.4
|
14.2
|
13.4
|
10.8
|
9.9
|
14.4
|
S. coni(n = 1)
|
17.4
|
15.2
|
14.9
|
10.8
|
10.5
|
9.5
|
15.7
|
S. simulans(n = 1)
|
20.7
|
17.2
|
14.4
|
12.5
|
11.6
|
11.4
|
14.5
|
S. equorum(n = 1)
|
19.3
|
17.7
|
15.2
|
12.1
|
10.7
|
10.2
|
17.6
|
ATCC 25923(n = 1)
|
24.4
|
19.4
|
16.7
|
14.8
|
12.7
|
10.7
|
18.3
|
Note: Inhibition zone > 16 mm indicate highly sensitivity; 11–15 mm indicate moderate sensitivity; 10 mm indicate light sensitivity; <10 mm indicate the resistance or ineffective. |
The MIC and MBC of six Tibetan herbal medicines were determined in comparison with Lonicera japonica against ten S. aureus isolates (Table 3). According to the results, five of six Tibetan herbal medicines showed certain antibacterial activity. Among them, the Lagotis brachystachy extract has the strongest antibacterial activity on S. aureus. The MIC and MBC values of Lagotis brachystachy were smaller than other Tibetan herbal medicines. The MIC values of Lagotis brachystachy, Aconitum flavum, Uncariarhync hophylla, Swertia bimaculata and Dracocephalu mtanguticum extracts were 1.95–7.8 mg/ml, 3.9–15.6 mg/ml, 7.8-31.25 mg/ml, 15.6–62.5 mg/ml and 15.6–62.5 mg/ml, respectively, and the corresponding MBC values were 7.8-31.25 mg/ml, 7.8–62.5 mg/ml, 31.25–125 mg/ml, 62.5–250 mg/ml and 62.5–250 mg/ml, respectively (Table 3).
Table 3
The MIC and MBC of six Tibetan medicinal plants against S. aureus
Isolates
|
MIC ( MBC) (mg/mL)
|
Lagotis
brachystachy
|
Aconitum
flavum
|
Uncaria
rhynchophylla
|
Swertia
bimaculata
|
Dracocephalum
tanguticum
|
Gentiana
urnula
|
Lonicera
japonica
|
S. aureus 1
|
1.95 (15.6)
|
3.9 (15.6)
|
15.6 (62.5)
|
31.25 (125)
|
31.25 (125)
|
125 (500)
|
7.8 (31.25)
|
S. aureus 2
|
7.8 (31.25)
|
15.6 (62.5)
|
31.25 (125)
|
62.5 (250)
|
62.5 (125)
|
250 (500)
|
31.25 (125)
|
S. aureus 3
|
3.9 (15.6)
|
3.9 (15.6)
|
7.8 (31.25)
|
15.6 (62.5)
|
31.25 (62.5)
|
62.5 (125)
|
7.8 (31.25)
|
S. aureus 4
|
3.9 (15.6)
|
7.8 (31.25)
|
15.6 (62.5)
|
31.25 (125)
|
31.25 (125)
|
62.5 (250)
|
7.8 (31.25)
|
S. aureus 5
|
3.9 (62.5)
|
7.8 (15.6)
|
15.6 (62.5)
|
62.5 (250)
|
62.5 (250)
|
125 (500)
|
15.6 (62.5)
|
S. aureus 6
|
3.9 (15.6)
|
3.9 (15.6)
|
7.8 (31.25)
|
31.25 (125)
|
31.25 (62.5)
|
125 (500)
|
31.25 (125)
|
S. aureus 7
|
7.8 (15.6)
|
7.8 (15.6)
|
7.8 (31.25)
|
31.25 (125)
|
31.25 (125)
|
62.5 (250)
|
7.8 (31.25)
|
S. aureus 8
|
3.9 (15.6)
|
3.9 (15.6))
|
15.6 (62.5)
|
31.25 (62.5)
|
31.25 (62.5)
|
250 (500)
|
15.6 (62.5)
|
S. aureus 9
|
3.9 (15.6)
|
7.8 (31.25)
|
15.6 (62.5)
|
62.5 (250)
|
62.5 (250)
|
250 (500)
|
7.8 (31.25)
|
ATCC 25923
|
1.95 (7.8)
|
3.9 (7.8)
|
7.8 (31.25)
|
15.6 (125)
|
15.6 (62.5)
|
62.5 (250)
|
7.8 (31.25)
|
Effect of water extracts of Tibetan herbal medicines on growth curve of S. aureus isolates
In the Kirby-Bauer disc diffusion and broth microdilution tests, the water extracts showed the varying degrees of effect against Staphylococcus isolates, indicating that the decoctions were activated as antimicrobial against the Staphylococcus isolates. The isolates in the negative control group showed a normal adaptation, exponential and stationary growth phases. Whereas the presence of water extracts of different Tibetan herbal medicines interfered with the culture growth of S. aureus isolates, delayed the beginning of the multiplication step, and prolonging the lag phase compared with the S. aureus isolate in negative control, except for the Gentiana urnul (Fig. 3A-F). The S. aureus isolates treated by water extracts of different Tibetan herbal medicines, in varying degrees, showed a reduction in cell density in the stationary phase according to the OD660 nm values, especially for the Lagotis brachystachy and Aconitum flavum, which had more than 80% reduction, while it has about 30% reduction for Gentiana urnul.